MSC-EVs role in myocardial protection after prolonged warm ischemia: preliminary data and creation of an effective protocol for ex-vivo heart perfusion

Background: To date, Donation after Brain and Circulatory Death (DBD and DCD) represent available sources of organs suitable for transplantation in humans. Heart donation and transplant, however, are not possible in Italy after DCD, as great concerns still exist regarding injury following the prolonged and inevitable warm ischemic time – Italian legislation requires at least 20 minutes of no-touch period – and further damage due to cold static storage and subsequent reperfusion, a damage known as Ischemia-Reperfusion Injury (IRI). Therefore, the development of effective strategies for myocardial protection during organ procurement, ex-vivo perfusion and implantation becomes crucial for an appropriate clinical translation. Aim of the study: To develop an effective protocol for ex-vivo heart perfusion, which will also enable organ rescuing and its function assessment during perfusion. Moreover, the protocol will include the administration of cellular particles, known as exosomes or extracellular vesicles (EVs), which for their properties have the potential to improve organ function. Methods: To achieve our goal, at first, a model of normothermic regional perfusion was created using a modified cardiopulmonary by-pass (CPB) circuit. Swine hearts are comparable for weight and anatomy to the humans one, and therefore, they were chosen for our tests. The circuit for ex-vivo heart perfusion resembled the features of other already available devices and it was composed by four peristaltic pumps, a heat exchanger and a membrane oxygenator, as for standard CPB. The heart was cannulated in a retrograde fashion with the aortic root to deliver solution into the coronary arteries and with the pulmonary trunk to get the blood back after perfusion. The whole system so made was then placed into a box where blood leaking from the heart was vented by further vent cannulas (one of them into the left ventricle to avoid distention). The circuit was primed with a solution consisting of half part animal blood and half part an extracellular crystalloid solution created ad hoc since perfusing the heart with whole blood optimal heart contraction was not achievable. The heart was then perfused with the priming and with a maintenance solution. Several attempts (n=8) were made, before finally reaching acceptable results. Cardiac function was assessed by the evidence of a beating heart. The capability of the system to maintain adequate perfusion of the organ (beating) over 2 hours and biochemical results (ions, lactate, and troponin) during perfusion were evaluated to verify the composition of the perfusate and to evaluate cardiac damage. Histological analyses were carried out to study microscopical changes into the myocardium. After standardizing the perfusion protocol 2 hearts underwent MSC-EVs administration. Results: In total 2 hearts, so far, underwent enriched perfusion with MSC-EVs. Our preliminary results show that our ex-vivo heart perfusion system is valid for the purpose and can be applicated to other experimental settings since the heart recovered part of its function. Troponin levels tend to increase later in heart treated with MSC-EVs. Histological proves show that there is no change in oedema, hemorrhage, ischemic injuries and cellular infiltration levels even with MSC-EVs. A difference was evidenced only in caspase 3 activation levels in heart treated with MSC-EVs. An important preliminary result is related to the morphological analysis of mitochondria, indeed TEM studies showed an increased preservation of these organelles, when the heart is treated with MSC-EVs. Conclusions: We achieved very preliminary proof of concept of the efficacy of the system and of the administration of the MSC-EVs in rescuing ischemic hearts. Nevertheless, the experiments are still ongoing and further data are necessary to improve the perfusion modalities and organ function assessment, before confirming the current data.